Cell membranes contain localized regions of specialized lipid and protein composition known as membrane microdomains. Recently, the properties of a class of membrane microdomains termed lipid rafts have become of great interest due to their proposed role in membrane trafficking, cell signaling, and the entry and exit of pathogens from cells. Commonly defined as cholesterol and sphingolipid-enriched domains, lipid rafts are thought to regulate protein function by concentrating some proteins into rafts, while segregating others in non-raft regions of the membrane. However, many of the fundamental properties of lipid rafts remain unknown, including the mechanism(s) by which specific proteins are targeted to lipid rafts. A commonly cited model for the association of proteins with lipid rafts is drawn by analogy to the partitioning of lipid probes in mixtures of liquid-ordered and liquid-disordered lipid phases. We hypothesize that this partitioning model is insufficient to explain the behavior of raft proteins in cells. Instead, we postulate that raft proteins are targeted to actively maintained domains in a cholesterol-dependent manner, and that the mode of membrane anchorage is a major determinant of the mechanism of raft association for any given protein. To test this hypothesis, we propose to investigate the mechanisms that govern the raft association of three commonly studied raft proteins: cholera toxin B-subunit (a glycolipid-binding protein), hemagglutinin (a transmembrane protein), and GFP-GPI (a glycosylphosphatidylinositol-anchored protein). To do so, we will perform biophysical measurements of raft- and non-raft proteins in living cells in combination with computer simulations of raft formation. The specific aims of these studies are (1) to determine if the sub-micron distribution and diffusional mobility of three representative raft proteins are consistent with an actively maintained model for raft formation;(2) to determine if different types of raft proteins co-localize or compete for residence in the same rafts;and (3) to generate in silico simulations of lipid raft assembly mechanisms. Completion of these studies will provide new information regarding the mechanisms that underlie targeting of proteins to rafts in cells. Such information is critical to the eventual design of therapies targeted at interfering with or enhancing lipid raft function to improve human health. These studies will also contribute to our long- term goal of understanding how membrane structure regulates cellular functions.